Peptide useful in immunomodulation

ABSTRACT

The present invention provides peptides and polynucleotides, and their use for immunomodulation, immunotherapy and vaccine particularly for anti-cancer therapy, and for diagnosis purposes. The immunomodulatory effect includes induction of proliferation and activation of peripheral blood lymphocytes and induction of an anti-tumor effect upon administration of peptides of the invention to subjects suffering from cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 11/411,832filed Apr. 27, 2006, now U.S. Pat. No. 7,329,639 B2, which is acontinuation of U.S. application Ser. No. 10/821,283 filed Apr. 9, 2004,now U.S. Pat. No. 7,122,372, which is a continuation of Internationalapplication PCT/IL02/00831 filed Oct. 15, 2002, the entire content ofeach of which is expressly incorporated herein by reference thereto.

FIELD OF THE INVENTION

The present invention relates to the field of immunotherapeutic vaccinesand particularly the identification and use of peptides recognized bythe immunomodulatory monoclonal antibodies designated BAT, topolynucleotides encoding these peptides, to pharmaceutical compositionscomprising the peptides or the polynucleotides and to use thereof inimmunomodulation, especially in anti-cancer therapy, and for diagnosticpurposes.

BACKGROUND OF THE INVENTION

Cancer in its different forms is a major cause of death in humans. Themost widely used therapeutic treatments of cancer are local therapy,such as surgery and radiation, or chemotherapy. The rapid increase ofknowledge in recent years about the molecular and cellular bases ofimmune regulation, particularly at the level of T-cell responses,provides a new arsenal of immunotherapeutic approaches including thedevelopment of tumor vaccines. Tumor vaccine is administered fortherapeutic or preventive purposes. This can include administration ofimmuno-potentiating agents as well as biological response modifiers suchas interferons and interleukins, in order to stimulate the immunesystem.

Vaccination with an antigen molecule, such as a peptide or a protein,generally leads to an antibody response or CD4+ helper T cell response(Raychaudhuri et al., 1993 Immunol Today 14:344). This immune responseis initiated by the binding of the antigen to selected majorhistocompatibility complex (MHC) molecules of either Class I or ClassII. The latter molecules are expressed primarily on cells involved ininitiating and sustaining immune responses such as T lymphocytes, Blymphocytes and macrophages. Class II molecules are recognized by CD4+helper T cell and induce their proliferation and the amplification ofthe immune response to the epitope that is displayed. Class I MHCmolecules are found on most nucleated cells and are recognized bycytotoxic T lymphocytes (CTLs) which destroy the antigen bearing cells.The CTL response is a major component of the immune system, active inimmune surveillance and destruction of infected or malignant cells andinvading organisms expressing foreign antigens on their surface. Theligand of the antigen-specific T lymphocyte receptor is a complex madeup of a peptide fragment of a foreign antigen 8 to 10 amino acids inlength, presented in the groove of MHC class I molecules. Unlike Bcells, T cells do not recognize intact native antigen molecules. Ingeneral, cytotoxic T cell activation requires that the antigen beprocessed endogenously and cleaved into specific peptide fragments whichare presented on the surface of antigen processing cells in associationwith class I MHC molecules.

Accordingly, a successful vaccine for cancer immunotherapy requires theidentification of a target antigen and the production of a cytotoxic Tcell response. Moreover, identification of cell surface antigensexpressed exclusively or preferentially on certain tumors allows theformation of selective treatment strategies.

Numerous disclosures exist concerning immunomodulatory peptides.WO94/20127 discloses means and methods for selecting immunogenicpeptides capable of specifically binding HLA-A2.1 allele and inducingT-cell activation. WO95/19783 relates to peptides based on an epitopederived from the product of the tumor associated gene MAGE-3. WO97/11715discloses a peptide which mimics MUCI or other cancer peptides.WO00/06723 discloses tumor specific antigen peptides and use thereof asanti-tumor vaccines. U.S. Pat. No. 6,406,700 discloses methods forisolating immunogenic complexes by using a cDNA library from cancer cellRNA.

International patent applications WO95/20605 and WO00/58363 which areincorporated herein by reference, describe a novel monoclonal antibodydesignated BAT-1, also designated herein BAT, which induces lymphocyteproliferation and cytolytic activity against tumor target cells. Asingle intravenous administration of BAT into mice bearing varioustumors resulted in striking anti-tumor effects manifested by regressionof tumors and prolongation of survival. BAT also induced regression ofhuman tumor xenografts transplanted into SCID mice that were engraftedwith human peripheral blood lymphocytes. The anti-tumor activity of BATis mediated by its immune stimulatory properties as was evident fromadoptive transfer experiments in which splenocytes from BAT treated miceinjected to mice bearing tumors induced regression of tumors. Themembrane determinant recognized by BAT has not yet been identified orcharacterized.

Several alternative methods of identifying the peptide epitopes bound bymonoclonal antibodies are recognized in the art. These methods includethe use of phage display libraries such as disclosed in U.S. Pat. Nos.5,223,409; 5,403,484; 5,571,698; 5,837,500 and continuations thereto.Phage display involves a selection technique enabling identification andisolation of a protein against a chosen target. The selection procedureis based on DNA molecules, each encoding a protein and a structuralsignal calling for the display of the protein on the outer surface of abacteriophage. The protein is expressed and the potential binding domainis displayed on the outer surface of the phage. The cells or virusesbearing the binding domains which recognize the target molecule areisolated, by a repetitive selection process called biopanning, andamplified. The successful binding domains are then characterized.

Epitope libraries can also be screened for epitope sequences which mimicthe epitope, i.e., sequences which do not identify a continuous linearnative sequence that necessarily occurs within a natural proteinsequence. These mimicking peptides are called mimotopes. In most casesmimotopes are short peptides which can be readily synthesized in largeamounts. Mimotopes of various binding sites have been found. Forexample, U.S. Pat. No. 5,877,155 provides an isolated peptide thatfunctionally mimics a binding site for a monoclonal antibody, themonoclonal antibody recognizing an epitope within the human plateletglycoprotein Ib/IX complex.

There is an unmet medical need for peptides capable of eliciting orstimulating an anti-tumor immune response in vivo.

SUMMARY OF THE INVENTION

The present invention now provides peptides that are useful asimmunomodulatory agents, for example, in stimulating immune responsesand in tumor growth inhibition. Thus, these peptides are useful intreatment of cancer and in treatment of autoimmune diseases.

In addition, the invention provides peptides that are recognized by BATmonoclonal antibody and, in particular, peptides that comprise epitopesor mimotopes recognized by the immunomodulatory antibody designatedBAT-1.

The invention also relates to a diagnostic agent and method fordiagnosing cancer in a subject. These can include an antibodyrecognizing at least one epitope encompassed within any of the peptidesof the invention.

The term ‘epitope’ referred to herein, relates to that part of anantigenic molecule that is recognized and bound by a T-cell receptor orby a B-cell receptor (i.e. a determinant on a large molecule againstwhich an antibody can be produced and to which it will bind). The termas used herein is intended to include antigenic determinants ofnaturally occurring molecules or synthetic molecules that can mimicnaturally occurring antigenic determinants. Molecules which mimic thenaturally occurring antigenic determinants may also be referred to as‘mimotopes’, and these terms may be used interchangeably in reference toepitopes which are not formed by a contiguous segment of the primarysequence of an antigen.

The peptides of the invention are recognized by the BAT-1 monoclonalantibody, also denoted herein as BAT, which is disclosed in WO95/20605and WO00/58363. The peptides of the invention are further recognized bygenetically modified antibodies which retain the biological activity ofBAT, including chimeras or CDR-grafted antibodies. A chimerichuman-mouse BAT antibody, containing mouse variable regions joined tohuman constant regions, is disclosed in WO00/58363.

It has previously been shown that BAT monoclonal antibody is beneficialin treating a variety of tumors including but not limited to: melanoma,lung carcinoma, prostate cancer, breast cancer, lymphomas and leukemias,colon carcinoma, and fibrosarcomas. The peptides and polynucleotides ofthe present invention are useful to elicit an immune response that willobviate the necessity to treat an individual with the antibodiesthemselves. Thus, the peptides and polynucleotides may be used to elicitantibodies that share the attributes of the previously known BATantibody.

In addition, the peptides of the invention are immunomodulatory. Thepeptides of the invention may serve as immunostimulatory agents toelicit anti-tumor activity or may serve as agents for immunotherapy oras immune-stimulators against infections, including in immunizationprocedures. Conversely, they can serve to inhibit undesirable immuneresponses such as immune responses that are involved in inflammatoryconditions, including but not limited to autoimmune diseases. Asimmunomodulators, these peptides can induce shifts in the immune systemfrom undesirable responses to beneficial responses. Thus, for examplethe peptides of the invention could be used to induce shifts from Thelper 1 (TH1) to T helper 2 (TH2) responses that have been postulatedto be of therapeutic value for suppressing or preventing autoimmunediseases or disorders.

There is thus provided, according to one embodiment of the invention, apeptide comprising at least one epitope recognized by a BAT monoclonalantibody, selected from:

(SEQ ID NO 1) Pro Arg Arg Ile Lys Pro Arg Lys Ile Met Leu Gln (SEQ ID NO2) Pro Arg Arg Ile Lys Pro Arg Lys Ile Met Leu Gln-amide (SEQ ID NO 3)Pro Arg Arg Phe Lys Pro Arg Lys Ile Asn/Asp Leu Gln (SEQ ID NO 4) ProArg Arg Ile Lys Pro Arg Lys Ile Asn/Asp Phe Gln (SEQ ID NO 5) Pro ArgArg Ile Lys Pro Arg Lys Ile Asn/Asp Leu Gln (SEQ ID NO 6) Pro Arg ArgIle Lys Ala Arg Lys Ile Met Leu Gln (SEQ ID NO 7) Pro Arg Lys Ile LysPro Arg Lys Ile Met Leu Gln (SEQ ID NO 8) -   -   Arg Ile Lys Pro ArgLys Ile Met Leu Gln (SEQ ID NO 9) Pro Arg Arg Ile Lys Pro Arg Lys IleMet -   - (SEQ ID NO 10) acetyl-Pro Arg Arg Ile Lys Pro Arg Lys Ile MetLeu Gln (SEQ ID NO 11) Gln Arg Ile Leu Gln Gln Ile Asn Leu Pro Arg Ile(SEQ ID NO 12) Gln Arg Ile Leu Gln Gln Ile Asn Leu Ala Arg Ile (SEQ IDNO 13) Gln Arg Ile Leu Gln Glu Ile Asn Leu Pro Arg Ile (SEQ ID NO 14)Gln Arg Ile Leu Gln Gln Ile Asn Leu Pro Lys Ile (SEQ ID NO 15)-   -   Ile Leu Gln Gln Ile Asn Leu Pro Arg Ile (SEQ ID NO 16) Gln ArgIle Leu Gln Gln Ile Asn Leu Pro -   - (SEQ ID NO 17) Asn Arg Ile Arg ThrAsn Thr Lys Leu Met Asn Ser

According to certain currently preferred embodiments, the presentinvention provides a peptide comprising at least one epitope recognizedby a BAT monoclonal antibody selected from SEQ ID NOs 1, 6, 8, 9, 10, 14and 16.

According to another particular embodiment, the present inventionprovides a peptide comprising at least one epitope recognized by a BATmonoclonal antibody, selected from: a peptide, a fragment of a peptide,a homolog, a variant, a derivative or a salt of a peptide having thesequence of any one of SEQ ID NOs 1 through 17, wherein the biologicalactivity of said peptides or fragments is retained.

According to yet another particular embodiment, the present inventionprovides a combination of any one of the peptides comprising at leastone epitope recognized by a BAT monoclonal antibody, selected from: apeptide, a fragment of a peptide, a homolog, a variant, a derivative ora salt of a peptide having the sequence of any one of SEQ ID NOs 1through 17, wherein the biological activity of said peptides orfragments is retained.

According to certain currently preferred embodiments, the presentinvention provides a peptide comprising at least one epitope recognizedby a BAT monoclonal antibody, selected from: a peptide, a fragment of apeptide, a homolog, a variant, a derivative or a salt of a peptidehaving the sequence of any one of SEQ ID NOs 1, 6, 8, 9, 10, 14, 16,wherein the biological activity of said peptides or fragments isretained.

According to yet another currently preferred embodiments, the presentinvention provides a combination of any one of the peptides comprisingat least one epitope recognized by a BAT monoclonal antibody, selectedfrom: a peptide, a fragment of a peptide, a homolog, a variant, aderivative or a salt of a peptide having the sequence of any one of SEQID NOs 1, 6, 8, 9, 10, 14, 16, wherein the biological activity of saidpeptides or fragments is retained.

According to yet another particular embodiment, the peptide of thepresent invention is capable of inhibiting binding of BAT monoclonalantibody to lymphoma cells, for example, to Daudi or Jurkat cells.

Furthermore, in another embodiment, the present invention provides apeptide useful for inhibiting tumor growth, selected from: a peptide, afragment of a peptide, a homolog, a variant, a derivative or a salt of apeptide having the sequence of any one of SEQ ID NOs 1 through 17,wherein the biological activity of said peptides or fragments isretained.

According to yet another particular embodiment, the present inventionprovides a combination of any one of the peptides useful for inhibitingtumor growth, selected from: a peptide, a fragment of a peptide, ahomolog, a variant, a derivative or a salt of a peptide having thesequence of any one of SEQ ID NOs 1 through 17, wherein the biologicalactivity of said peptides or fragments is retained.

In certain currently preferred embodiments, the present inventionprovides a peptide useful for inhibiting tumor growth, selected from: apeptide, a fragment of a peptide, a homolog, a variant, a derivative ora salt of a peptide having the sequence of any one of SEQ ID NOs 1, 6,8, 9, 10, 14, 16, wherein the biological activity of said peptides orfragments is retained.

According to another certain currently preferred embodiments, thepresent invention provides a combination of any one of the peptidesuseful for inhibiting tumor growth, selected from: a peptide, a fragmentof a peptide, a homolog, a variant, a derivative or a salt of a peptidehaving the sequence of any one of SEQ ID NOs 1, 6, 8, 9, 10, 14, 16,wherein the biological activity of said peptides or fragments isretained.

In another embodiment, the present invention provides a peptide capableof inducing an immune response against tumor cells, selected from: apeptide, a fragment of a peptide, a homolog, a variant, a derivative ora salt of a peptide having the sequence of any one of SEQ ID NOs 1through 17, wherein the biological activity of said peptides orfragments is retained.

According to yet another particular embodiment, the present inventionprovides a combination of any one of the peptides capable of inducing animmune response against tumor cells, selected from: a peptide, afragment of a peptide, a homolog, a variant, a derivative or a salt of apeptide having the sequence of any one of SEQ ID NOs 1 through 17,wherein the biological activity of said peptides or fragments isretained.

In certain currently preferred embodiments, the present inventionprovides a peptide capable of inducing an immune response against tumorcells, selected from: a peptide, a fragment of a peptide, a homolog, avariant, a derivative or a salt of a peptide having the sequence of anyone of SEQ ID NOs 1, 6, 8, 9, 10, 14, 16, wherein the biologicalactivity of said peptides or fragments is retained.

According to currently preferred embodiments, the present inventionprovides a combination of any one of the peptides capable of inducing animmune response against tumor cells, selected from: a peptide, afragment of a peptide, a homolog, a variant, a derivative or a salt of apeptide having the sequence of any one of SEQ ID NOs 1, 6, 8, 9, 10, 14,16, wherein the biological activity of said peptides or fragments isretained.

Further provided according to another embodiment of the invention, is apolynucleotide encoding at least one peptide recognized by BATmonoclonal antibodies. The nucleic acid sequences include both the DNAsequence that is transcribed into RNA and the RNA sequence that istranslated into a peptide.

According to certain embodiments of the present invention, thepolynucleotide comprises a sequence selected from the group consistingof:

(SEQ ID NO 18) CCTCGACGAATAAAGCCCAGGAAGATCATGCTGCAA (SEQ ID NO 26)CAGAGGATACTGCAGCAAATTAATCTTCCCAGGATC (SEQ ID NO 32)AACCGAATCAGGACAAATACTAAGCTCATGAACAGC

According to other embodiments, the polynucleotides of the invention areinferred from the amino acid sequence of the peptides of the invention.As is known in the art several alternative polynucleotides are possibledue to redundant codons, while retaining the biological activity of thetranslated peptides.

Thus, according to further embodiments, the polynucleotide of theinvention comprise a sequences selected from:

(SEQ ID NO 19) CCTCGACGATTYAAGCCCAGGAAGATCRAYCTGCAA (SEQ ID NO 20)CCTCGACGAATAAAGCCCAGGAAGATCRAYTTYCAA (SEQ ID NO 21)CCTCGACGAATAAAGCCCAGGAAGATCRAYCTGCAA (SEQ ID NO 22)CCTCGACGAATAAAGGCXAGGAAGATCATGCTGCAA (SEQ ID NO 23)CCTCGAAAYATAAAGCCCAGGAAGATCATGCTGCAA (SEQ ID NO 24)CGAATAAAGCCCAGGAAGATCATGCTGCAA (SEQ ID NO 25)CCTCGACGAATAAAGCCCAGGAAGATCATG (SEQ ID NO 27)CAGAGGATACTGCAGCAAATTAATCTTGCXAGGATC (SEQ ID NO 28)CAGAGGATACTGCAGGARATTAATCTTCCCAGGATC (SEQ ID NO 29)CAGAGGATACTGCAGCAAATTAATCTTCCCAAYATC (SEQ ID NO 30)ATACTGCAGCAAATTAATCTTCCCAGGATC (SEQ ID NO 31)CAGAGGATACTGCAGCAAATTAATCTTCCCwherein Y=T or C; R=A or G; X=T or C or A or G)

The nomenclature used to describe peptide and polynucleotide compoundsof the invention follows the conventional practice wherein the aminogroup (N-terminus) and the 5′ are presented to the left and the carboxylgroup (C-terminus) and 3′ to the right.

It is to be understood explicitly that the scope of the presentinvention encompasses homologs, analogs, variants, derivatives andsalts, including shorter and longer peptides and polynucleotides, aswell as peptide and polynucleotide analogs with one or more amino acidor nucleic acid substitution, as well as amino acid or nucleic acidderivatives, non-natural amino or nucleic acids and synthetic amino ornucleic acids as are known in the art, with the stipulation that thesemodifications must preserve the biological activity of the originalmolecule. Specifically any active fragments of the active peptides aswell as extensions, conjugates and mixtures are disclosed according tothe principles of the present invention.

Also provided, according to another embodiment of the invention, is aconstruct comprising a polynucleotide encoding for at least one peptiderecognized by a BAT monoclonal antibody.

Furthermore, in another embodiment, the present invention provides avector, for example, a plasmid or a virus, comprising a polynucleotideencoding at least one peptide recognized by a BAT monoclonal antibody.

Further provided, according to other embodiment of the invention, is ahost cell comprising a polynucleotide encoding at least one peptiderecognized by the BAT monoclonal antibody.

In one embodiment the host cell is capable of expressing at least oneepitope recognized by BAT monoclonal antibody.

In yet another embodiment, the present invention provides a method and acomposition for treating cancer in a subject in need thereof, comprisingthe step of administering to a patient a therapeutically effectiveamount of a pharmaceutical composition of the present invention.

In one embodiment the pharmaceutical composition comprises apharmaceutical carrier and an active ingredient, which is a peptideaccording to embodiments of the invention.

In another embodiment the pharmaceutical composition comprises apharmaceutical carrier and an active ingredient, which is apolynucleotide encoding at least one peptide recognized by a BATmonoclonal antibody.

In one embodiment the polynucleotide comprises a sequence selected fromSEQ ID NOs 18 through 31.

In currently preferred embodiments the polynucleotide comprises asequence selected from SEQ ID NOs 18, 22, 24, 25, 29, 31.

Also provided according to an embodiment of the invention is animmunomodulatory vaccine comprising a pharmaceutically acceptableadjuvant, selected from the group of an aluminum salt and an oilemulsion, and at least one peptide recognized by a BAT monoclonalantibody.

In one embodiment, the peptide comprises a sequence selected from SEQ IDNOs 1 to 17.

The term “vaccine” or “vaccination” referred to herein relates to amodality or process that induces modulation of the immune system, forexample, but not limited to, a composition or process that induce theactivation of T-lymphocytes or that induces the production ofantibodies.

According to another embodiment of the invention there is provided adiagnostic agent for detecting the presence of tumor cells comprising apeptide, a fragment of a peptide, a homolog, a variant, a derivative ora salt of a peptide having the sequence of any one of SEQ ID NOs 1through 17, wherein the biological activity of said peptides orfragments is retained.

According to yet another particular embodiment, the present inventionprovides a diagnostic agent for detecting the presence of tumor cellscomprising a combination of any one of the peptides, a fragment of apeptide, a homolog, a variant, a derivative or a salt of a peptidehaving the sequence of any one of SEQ ID NOs 1 through 17, wherein thebiological activity of said peptides or fragments is retained.

In certain currently preferred embodiments, the peptide comprises thesequence of any one of SEQ ID NOs 1, 6, 8, 9, 10, 14, and 16.

Furthermore, in another embodiment the present invention provides amethod for diagnosing cancer comprising the steps of: contacting asample from a subject with a peptide recognized by a BAT monoclonalantibody; determining the extent of binding of said peptide to thesample; and comparing the extent of binding of said peptide to thesample with a known control (such as a predetermined calibration scale)thereby obtaining information regarding the occurrence of cancer in thesample.

Furthermore, in another embodiment the present invention provides amethod for diagnosing an inflammatory disorder or autoimmune diseasecomprising the steps of: obtaining a sample of immune cells from anindividual; exposing said immune cells to at least one peptiderecognized by a BAT monoclonal antibody; and monitoring the response ofsaid immune cells compared to the response of said immune cells notexposed to the peptide.

According to another embodiment, the present invention provides anantibody recognizing at least one epitope selected from: a peptide, afragment of a peptide, a homolog, a variant, a derivative or a salt of apeptide having the sequence of any one of SEQ ID NOs 1 through 17,wherein the biological activity of said peptides or fragments isretained.

According to currently preferred embodiments, the present inventionprovides an antibody a combination of any one of the peptides comprisingat least one epitope recognized by a BAT monoclonal antibody, selectedfrom: a peptide, a fragment of a peptide, a homolog, a variant, aderivative or a salt of a peptide having the sequence of any one of SEQID NOs 1 through 17, wherein the biological activity of said peptides orfragments is retained.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description and appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibition of BAT monoclonal antibody binding toimmobilized phage bearing the insert of SEQ ID NO 1 (Peptide A) by thefree peptide.

FIG. 2 shows the results of a flow cytometry experiment in which PeptideA inhibition of BAT mAb binding to B-lymphoblastoid cells wasdetermined.

FIG. 3 is a graph showing growth inhibition of melanoma s.c. tumord, byimmunization with the synthetic Peptide A.

FIG. 4 is a graph showing binding of sera from Peptide A immunized miceto Peptide A coated plates.

FIG. 5 shows the binding of BAT monoclonal antibody to selectedpeptides, from the phage-library, in the second experiment.

FIG. 6 presents growth inhibition of lung tumors by Peptide A and itsanalogues.

FIG. 7 presents growth inhibition of lung tumors by Peptide B and itsanalogues.

FIGS. 8A, 8B and 8C exhibit cytolytic activity in splenocytes of miceimmunized with Peptides A and B.

FIGS. 9A-9D show inhibition of BAT monoclonal antibody binding to Daudicells by anti-Peptides A and B antibodies.

FIG. 10 presents induction of human lymphocyte proliferation byanti-Peptides A and B antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention concerns peptides, polynucleotides andpharmaceutical and vaccine compositions including same which are usefulin modulation of immune responses, in inhibition of tumor growth, bothprimary tumor and metastases, in treatment of cancer and of inflammatorydisorders including autoimmune diseases by way of prevention and cure.Specifically, the present invention provides vaccines which includepeptides recognized by the immune modulator BAT monoclonal antibodywhich is disclosed in the above-mentioned International PatentApplications WO 95/20605 and WO 00/58363.

a. Preferred Modes for Carrying Out the Invention

According to a first aspect of the present invention there are providedpeptides recognized by BAT monoclonal antibody. Several methods foridentifying peptides that are capable of binding to the BAT antibody areknown in the art. For example, raising peptides against the BAT antibodywhereas the peptides may be selected from peptide libraries, such as,bacteriophage library, chemical library, hybridoma cell library; cellspreferably B lymphocytes and T cell and more preferably Daudi cells andJurkat cells; chemical syntheses that might produce a set or a subset ofmolecules having high affinity for the sequence of BAT monoclonalantibody; designing molecules intended to have a high affinity for BATsequences using computer-assisted or other theoretical approaches; usingin-vitro evolution of nucleic acids capable of binding to the sequenceof BAT.

According to preferred embodiments of the present invention a phageepitope library is employed in order to identify peptides that bind BATmonoclonal antibody and to prepare an immunotherapy modality. Mostpreferably, a 12-mer-phage peptide library constructed using phage M13is used to identify peptides as the putative antibody binding epitopeand consequently to identify the polynucleotide sequence encoding thepeptides.

Although the peptide will preferably be substantially free of othernaturally occurring host cell proteins or protein fragments, in someembodiments the peptides can be synthetically conjugated to nativefragments or particles.

According to a certain preferred embodiments, the 12-mer peptides of thepresent invention are:

-   SEQ ID NO 1, also termed hereinafter Peptide A: Pro Arg Arg Ile Lys    Pro Arg Lys Ile Met Leu Gln-   SEQ ID NO 11, also termed hereinafter Peptide B: Gln Arg Ile Leu Gln    Gln Ile Asn Leu Pro Arg Ile-   SEQ ID NO 17, also termed hereinafter Peptide C: Asn Arg Ile Arg Thr    Asn Thr Lys Leu Met Asn Ser

According to the principles of the present invention the scope of theinvention further includes any variants, derivative or salts of theabove peptides.

The term “variant” as used herein refers to a peptide sequence thatpossesses at least one modified structural property compared to theoriginal peptide while substantially retaining the biological activityof the resulting peptide as compared with the original peptide or theBAT monoclonal antibody. For example, one or more of the amino acidresidues of the original peptides are replaced by different amino acidresidues, or are deleted, or one or more amino acid residues are addedto the original peptides, a peptide bond is modified, cyclization isintroduced to the structure of the original peptide, a backbone ismodified. A variant may have altered binding to BAT monoclonal antibodythan the original peptide. A variant may have at least 70% identity withthe original peptide, preferably 80% or 90% identity. A variant may alsobe referred to as ‘analog’ or ‘homolog’, and these terms may be usedinterchangeably.

In one embodiment, the present invention comprises a salt of peptide ofthe invention. The term “salt” includes acid addition salts which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. The term also includes base addition salts whichare formed from inorganic bases such as, for example, sodium, potassium,ammonium, and calcium, and from organic bases such as isopropylamine,trimethylamine, histidine, and the like.

In another embodiment the present invention comprises a derivative of apeptide of the invention. The term “derivative” of a peptide as usedherein refers to a peptide that contains additional chemical moietiesnot normally a part of the peptide. Such modifications may be introducedinto the molecule by reacting targeted amino acid residues of thepeptide with an organic derivatizing agent that is capable of reactingwith selected side chains or terminal residues. Examples of chemicalderivatives, by way of illustration and not by way of limitation,include a peptide in which the C-terminus or the N-terminus of thepeptides of the present invention, or both, are substituted with acarboxylic acid protecting group or an amine protecting group,respectively. Suitable protecting groups are described in Green andWuts, “Protecting Groups in organic Synthesis”, John Wiley and Sons,Chapters 5 and 7, 1991. Examples of N-terminal protecting groups includeacyl groups (—CO—R₁) and alkoxy carbonyl or aryloxy carbonyl groups(—CO—O—R₁), wherein R₁ is an aliphatic, substituted aliphatic, benzyl,substituted benzyl, aromatic or a substituted aromatic group. Examplesof acyl groups include acetyl, (ethyl)-CO—, n-propyl-CO—,iso-propyl-CO—, n-butyl-CO—, sec-butyl-CO—, t-butyl-CO— and phenyl-CO—.Examples of alkoxy carbonyl and aryloxy carbonyl groups includeCH₃—O—CO—, (ethyl)-O—CO—, n-propyl-O—CO—, iso-propyl-O—CO—,n-butyl-O—CO—, sec-butyl-O—CO—, t-butyl-O—CO—, phenyl-O—CO—. Thecarboxyl group at the C-terminus can be protected, for example, as anamide (i.e., the hydroxyl group at the C-terminus is replaced with —NH₂,—NHR₂ and —NR₂R₃) or ester (i.e. the hydroxyl group at the C-terminus isreplace with —OR₂). R₂ and R₃ are independently an aliphatic,substituted aliphatic, benzyl, substituted benzyl, aryl or a substitutedaryl group.

A variant, derivative or salt of a peptide of the invention may beadvantageous, as compared to the original peptide, if they possess atleast one of the following properties: improved solubility, prolongedduration of action, superior biological activity, increased stability todegradation by enzymes and therefore increased in vivo half lives,eliminated or attenuated undesirable side effects and the like.

The term “biological activity” as used herein, includes at least one ofthe following activities: induction of anti-tumor effect, modificationof immune response, stimulation of immune response against tumor cells,inhibition of the binding of BAT monoclonal antibody to lymphomacells—particularly Daudi or Jurkat cells, detection of cancer, detectionof an inflammatory disorder or autoimmune disease or, prevention oftumor development.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,and antibodies of the invention in prevention of the occurrence of tumorin the first place.

The peptide or variant may be generated through recombinant DNAtechnologies, well known to those skilled in the art, following cloninginto an expression vector, transfection into host cells and the proteinharvesting according to methods known in the art. In a preferredembodiment the peptides and variants are prepared by known peptidesynthesis techniques.

Currently preferred embodiments of the invention are variants obtainedby conservative modifications introduced to SEQ ID NO 1 (Peptide A).Currently most preferred embodiments include the following variants:

(SEQ ID NO 2) Pro Arg Arg Ile Lys Pro Arg Lys Ile Met Leu Gln-amide (SEQID NO 3) Pro Arg Arg Phe Lys Pro Arg Lys Ile Asn/Asp Leu Gln (SEQ ID NO4) Pro Arg Arg Ile Lys Pro Arg Lys Ile Asn/Asp Phe Gln (SEQ ID NO 5) ProArg Arg Ile Lys Pro Arg Lys Ile Asn/Asp Leu Gln (SEQ ID NO 6) Pro ArgArg Ile Lys Ala Arg Lys Ile Met Leu Gln (SEQ ID NO 7) Pro Arg Lys IleLys Pro Arg Lys Ile Met Leu Gln (SEQ ID NO 8) -   -   Arg Ile Lys ProArg Lys Ile Met Leu Gln (SEQ ID NO 9) Pro Arg Arg Ile Lys Pro Arg LysIle Met -   - (SEQ ID NO 10) acetyl-Pro Arg Arg Ile Lys Pro Arg Lys IleMet Leu Gln

Additional currently preferred embodiments of the invention are variantsobtained by conservative modifications introduced to SEQ ID NO 11(Peptide B). Currently most preferred embodiments include the followingvariants:

(SEQ ID NO 12) Gln Arg Ile Leu Gln Gln Ile Asn Leu Ala Arg Ile (SEQ IDNO 13) Gln Arg Ile Leu Gln Glu Ile Asn Leu Pro Arg Ile (SEQ ID NO 14)Gln Arg Ile Leu Gln Gln Ile Asn Leu Pro Lys Ile (SEQ ID NO 15)-   -   Ile Leu Gln Gln Ile Asn Leu Pro Arg Ile (SEQ ID NO 16) Gln ArgIle Leu Gln Gln Ile Asn Leu Pro -   -

The biological activity of the variants is monitored and compared withthat of the selected peptides and of the BAT monoclonal antibody asexemplified hereinbelow.

Without wishing to be bound by any theory, it is possible that thepeptides of the invention elicit an immune response similar to thatobtained by the BAT monoclonal antibody.

Thus, preferred methods for monitoring the biological activity of thepeptides and the variants include: competitive inhibition with BATmonoclonal antibody to determinants recognized by BAT, e.g. Daudi cellsand Jurkat cells; competitive inhibition to determinants recognized byBAT, e.g. Daudi cells and Jurkat cells, between sera of immunizedsubjects and BAT monoclonal antibody; immunizing with the peptidestumor-bearing animals and monitoring tumor growth; immunizing with thepeptides naive animals and monitoring tumor development upon inductionof cancer; monitoring immune response upon immunization, preferablymonitoring the cytolytic activity of NK and CTL cells upon immunizationwith the peptides; monitoring lymphocyte proliferation upon immunizationwith anti-peptides antibodies.

According to a second aspect of the present invention there is provideda construct comprising a polynucleotide encoding for the peptidesrecognized by the BAT monoclonal antibody.

In several preferred embodiments the polynucleotides of the inventionare isolated from the phage clones expressing peptides recognized by BATmonoclonal antibody, and are selected from: SEQ ID NO 18, encoding thepeptide of SEQ ID NOS 1 (Peptide A), 2 and 10; SEQ ID NO 26 encoding thepeptide of SEQ ID NO 11 (Peptide B); SEQ ID NO 32 encoding the peptideof SEQ ID NO 17 (Peptide C).

According to other general embodiments, the polynucleotides of theinvention are derived from the amino acid sequence of the peptides ofthe invention, including variations due to redundant codons providingthat the biological activity of the translated peptide is retained.

Thus, according to further embodiments the polynucleotide of theinvention are selected from the group of sequences consisting of:

-   SEQ ID NO 19 encoding the peptide of SEQ ID NO 3;-   SEQ ID NO 20 encoding the peptide of SEQ ID NO 4;-   SEQ ID NO 21 encoding the peptide of SEQ ID NO 5;-   SEQ ID NO 22 encoding the peptide of SEQ ID NO 6;-   SEQ ID NO 23 encoding the peptide of SEQ ID NO 7;-   SEQ ID NO 24 encoding the peptide of SEQ ID NO 8;-   SEQ ID NO 25 encoding the peptide of SEQ ID NO 9;-   SEQ ID NO 26 encoding the peptide of SEQ ID NO 10;-   SEQ ID NO 27 encoding the peptide of SEQ ID NO 12;-   SEQ ID NO 28 encoding the peptide of SEQ ID NO 13;-   SEQ ID NO 29 encoding the peptide of SEQ ID NO 14;-   SEQ ID NO 30 encoding the peptide of SEQ ID NO 15;-   SEQ ID NO 31 encoding the peptide of SEQ ID NO 16;

The constructs comprising the polynucleotides encoding the peptides ofthe invention may further include promoters, enhancers and otherregulatory sequences necessary for expression, transcription andtranslation, as are well known in the art. The constructs can be furtherprovided with appropriate linkers and can be ligated into expressionvectors available in the art. The vectors can be used to transformsuitable hosts to produce the desired peptides. Vectors may includerestriction enzyme sites for the insertion of additional genes and forselection markers, as well as elements necessary for propagation andmaintenance of vectors within cells.

Cells presenting peptides recognized by the monoclonal antibody BAT arealso included in the present invention, for instance, antigen presentingcells including dendritic cells or macrophages which are also known asuseful immunogens.

An antibody recognizing an epitope within any one of SEQ ID NOs 1through 17 is further provided in the present invention.

The term “antibody” is used herein in the broadest sense and coversmonoclonal antibodies (including full length monoclonal antibodies) andantibody fragments so long as they exhibit the desired biologicalactivity. “Antibody fragments” comprise a portion of a full lengthantibody, generally the antigen binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.

Methods for the production of antibodies are well known in the arts,including eliciting antibodies from the sera of subjects immunized withthe target determinant of said antibodies, as well as recombinanttechniques.

b. Pharmacology

The present invention further relates to a pharmaceutical compositionwhich includes at least one epitope recognized by the BAT monoclonalantibody as the pharmaceutically active ingredient. The pharmaceuticallyactive ingredient is selected from the group containing: a peptidecomprising at least one epitope, a polynucleotide encoding at least oneepitope, a polynucleotide encoding at least one of said peptide, aconstruct comprising at least one of said polynucleotide, a vectorcomprising at least one of said construct.

The peptide of the present invention, or a pharmacologically acceptablesalt thereof may be mixed with an excipient, carrier, diluent, andoptionally, a preservative or the like, pharmacologically acceptablevehicles as known in the art. Examples of excipients include, glucose,mannitol, inositol, sucrose, lactose, fructose, starch, cornstarch,microcrystalline cellulose, hydroxypropylcellulose,hydroxypropyl-methylcellulose, polyvinylpyrrolidone and the like.Optionally, a thickener may be added, such as a natural gum, a cellulosederivative, an acrylic or vinyl polymer, or the like. The pharmaceuticalcomposition including the peptide may further comprise a biodegradablepolymer selected from poly-1,4-butylene succinate, poly-2,3-butylenesuccinate, poly-1,4-butylene fumarate and poly-2,3-butylene succinate,incorporating the peptide of the invention as the pamoate, tannate,stearate or palmitate thereof. Such compositions are known in the art asdescribed, for example, in U.S. Pat. No. 5,439,688.

The pharmaceutically active ingredient of the composition may beconjugated to a matrix or to a proteinaceous carrier, for exampletetanus or diphtheria toxoids or oxidized KLH, in order to stimulate Tcell help, or to other immuno-potentiating agents as well as biologicalresponse modifiers such as interferons, interleukins etc., in order tostimulate the immune system. The compositions for administration tohumans may further comprise adjuvants that are suitable for human use,such as alum, which is approved for human use, or submicron emulsionsthat are intended for human use as disclosed for example in WO95/11700.Appropriate ranges of ingredients for preparing compositions with orwithout additional diluents, carriers or adjuvants are known in the art.

The preparation of pharmaceutical compositions comprising peptides iswell known in the art, as disclosed for example in U.S. Pat. Nos.5,736,519, 5,733,877, 5,418,219, 5,354,900, 5,298,246, 5,164,372,4,900,549 and 4,457,917. Means for processing the pharmaceuticalcompositions of the present invention include, without limitations,conventional mixing, dissolving, granulating, grinding, pulverizing,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

According to yet another aspect of the present invention a peptidevaccine is provided. The two major types of peptide vaccine are:peptides mixed with adjuvant substances and peptides which areintroduced together with an antigen presenting cell (APC; Mayordomo etal., Nature Med. 1:1297, 1995). The most common cells used for thelatter type of vaccine bone marrow and peripheral blood deriveddendritic cells, as this cells express costimulatory molecules that helpactivation of CTL. WO00/06723 discloses a cellular vaccine compositionwhich includes an antigen presenting cell presenting tumor associatedantigen peptides. Presenting the peptide can be effected by loading theAPC with a polynucleotide (e.g., DNA, RNA) encoding the peptide orloading the APC with the peptide itself.

In accordance with the first type of peptide vaccine, adjuvantsubstances that stimulate immunogenicity are mixed with the peptide inorder to improve the immune response to the peptide. Immunologicaladjuvants have generally been divided into two basic types: aluminumsalts and oil emulsions. Aluminum phosphate and hydroxide (alum)adjuvants induce elevated levels of antibody against antigens inalum-based vaccines above those obtained with the corresponding aqueousvaccine. Numerous alum-based vaccines, including methods of preparationthereof, were developed as, for example, disclosed in U.S. Pat. Nos.5,747,653, 6,013,264, 6,306,404 and 6,372,223. However, aluminumcompounds have not always enhanced the immunogenicity of vaccines.

The main components of the oil-based adjuvants are: oil, emulsifier andimmunostimulant. The earliest types of emulsified oil-based adjuvantsare Incomplete Freund's Adjuvant (IFA), consisting of an approximately50:50 water-in-oil emulsion, and complete Freund's adjuvant (CFA), asimilar preparation with inclusion of killed mycobacteria. The powerfulantibody-stimulating effect of CFA has not been surpassed by any otheradjuvant. However, because of severe toxic reactions CFA can be usedonly for experimental purposes and not in human or veterinary vaccines.The use of IFA in humans has been limited to those clinical situationsin which aqueous vaccines are relatively impotent and aluminum compoundshave not provided enough adjuvant activity. Example of improvedemulsions as vaccine adjuvants, by enhancing the immunogenicity of theantigen, include submicron emulsions as disclosed in U.S. Pat. No.5,961,970 and solid fat nanoemulsions as disclosed in U.S. Pat. No.5,716,637 for example.

Preferred means for administering peptides are through intravenous,intramuscular or subcutaneous administration. Oral administration isexpected to be less effective, because a peptide may be digested beforebeing taken up. Decomposition in the digestive tract may be lessened byuse of certain compositions, for instance, by confining the peptide ofthe invention in microcapsules such as liposomes. The pharmaceuticalcomposition of the invention may also be administered to other mucousmembranes. The pharmaceutical composition is then provided in the formof a suppository, nasal spray or sublingual tablet.

The uptake of a peptide of the invention may be facilitated by a numberof methods. For instance, a non-toxic derivative of the cholera toxin Bsubunit, or of the structurally related subunit B of the heal-labileenterotoxin of enterotoxic Eschericia coli may be added to thecomposition, as disclosed in U.S. Pat. No. 5,554,378.

The peptides according to an embodiment of the invention may be alsoadministered via liposomes, slow releasing particles and the like, asknown in the art, so as to increase the immunogenicity of the peptides.

A composition according to an embodiment of the invention can bedirectly administered to an individual for immunizing the individual.Alternatively, in accordance with an embodiment of the invention, thepeptides may be used to generate new antibodies with the attribute andactivities of known BAT monoclonal antibodies. Ex-vivo activation ofT-cells by these peptides may also elicit the desired activity ofimmunostimulation. Thus, the composition can be used for inducingantibodies in an ex-vivo system and the induced antibodies can then beadministered to an individual for treating an autoimmune disease, aninfection or cancer. The composition can also be used in an ex-vivosystem to stimulate T-cells to be administered in a process of adoptiveimmunotherapy, as described in the art.

The present invention also includes the use of the peptide orpolynucleotide as a diagnostic agent for diagnosing cancer in anindividual. According to an embodiment of the invention a sample from aindividual, such as a blood sample or a sample from a patient's GI tractfluids, or cerebrospinal fluid or any other relevant sample can becontacted, in vivo or in vitro, with a peptide or polynucleotideaccording to the invention and the extent of binding of the peptide orpolynucleotide to the sample can be determined, such as by ELISA, so asto provide information regarding the occurrence of BAT in the sample.For example antibodies to the peptides of the invention may be used todiagnose cancer or to monitor its progression if present in a body fluidespecially serum or plasma. Conversely, in the patient's lymph nodesespecially in draining lymph nodes in proximity to a suspected tumor, itmay be possible to screen the T cells using the peptides of theinvention.

The present invention provides methods for the treatment of autoimmunediseases, cancer and for anti-cancer vaccination. Pharmaceuticalcompositions suitable for use in context of the present inventioninclude compositions wherein the active ingredients are contained in anamount effective to achieve the intended purpose. All formulations foradministration should be in dosages suitable for the chosen route ofadministration. More specifically, a “therapeutically effective” dosemeans an amount of a compound effective to prevent, alleviate orameliorate symptoms of a disease of the subject being treated.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

Toxicity and therapeutic efficacy of the compositions described hereincan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., by determining the IC₅₀ (theconcentration which provides 50% inhibition) and the maximal tolerateddose for a subject compound. The data obtained from these cell cultureassays and animal studies can be used in formulating a range of dosagefor use in human. The dosage may vary depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. Depending onthe severity and responsiveness of the condition to be treated, dosingcan also be a single administration of a slow release composition, withcourse of treatment lasting from several days to several weeks or untilcure is effected or diminution of the disease state is achieved. Theamount of a composition to be administered will, of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration, the judgment of the prescribing physician, and allother relevant factors.

EXAMPLES

The present invention will be further described and exemplified by thefollowing non-limitative examples.

Example 1 Preparation of BAT Monoclonal Antibody

BAT was generated, purified and characterized as previously disclosed inWO 95/20605, WO 00/58363 and subsequent publications. In brief, BALB-Cmice were immunized with membranes of Daudi cells. Spleen cells werefused with myeloma NSO cells. Clones producing BAT were selected by theability of supernatants to bind Daudi cells and to induce proliferationof peripheral blood mononuclear cells. Hybridoma cells were grown inRPMI 1640 supplemented with 10% fetal calf serum (for Experiment 1 inExample 2) or with PFHM serum free protein media (GIBCO; for Experiment2 in Example 2), sodium pyruvate, glutamine, and antibiotics andincubated at 37° C. in a humidified atmosphere containing 5% CO₂. BATwas purified on a protein G Sepharose column according to manufacturer'sinstructions (Pharmacia Corp. NJ, USA). Biotinylation, was performed asfollows: 100 μg of the immunoglobulin fraction of BAT monoclonalantibody in 1 ml of 0.1 M NaHCO3, pH 8.6, was incubated for 2 hours atroom temperature with 5 μg of biotin amidocoproate N-hydroxysuccinimideester (SIGMA, Israel) from a stock solution of 1 mg/ml indimethylformamide and dialyzed at 4° C. against phosphate bufferedsaline pH 7.4.

Example 2 Isolation of Peptides by Phage-Display Analysis

Experiment 1

Materials and Methods

a. Isolation of Epitope-Presenting Phage from a Phage Epitope Library

The phage display peptide library was based on a combinatorial libraryof random 12-mer peptides fused to a minor coat protein (pIII) of M13phage. A library sample containing 4×10¹⁰ infectious phage particles wassubjected to 6 rounds of panning and amplification. For each selectioncycle 100 μg of biotinylated BAT monoclonal antibody was used. The phagewas pre-incubated with the biotinylated antibody at room temperature for1 hour. The reaction mixtures were then layered in 1 ml of TBS 0.5%Tween on streptavidin coated and blocked 60-mm polystyrene Petri dishesfor 30 minutes at room temperature. Unbound phages were removed by 10times washings in TBS 0.5% Tween. The remaining phages were eluted with1 ml 0.2M Glycine-HCl (pH 2.2), 1 mg/ml BSA. The eluate was neutralizedand used to infect E. coli stain ER2537. After each round of panningphage was titer on LB/IPTG/Xgal plates. The unamplified last round wastitered and plaques were used for sequencing.

b. Phage-ELISA

Wells of microtiter plates were coated with 100 μl of a 1:1000 dilution(0.1M NaHCO3, pH 8.6) of rabbit anti-phage M13 serum by incubationovernight at 4° C. Coated plates were washed 3 times with PBS 0.05%Tween 20 and 100 μl of enriched phage clones, containing 10⁹ phageparticles, were then added to the wells and incubated for 1 hour at 37°C. Wells were blocked with 1% BSA in PBS for 1 hour at room temperature,washed and incubated with the antibody overnight at 4° C. For inhibitionexperiments, peptides were pre-incubated with the antibody for 30minutes, before their addition to phage coated wells. After washing,bound antibody was detected by incubation with anti-mouse IgG Peroxidaseconjugated (Fab specific) for 45 minutes. After washing with o-phenylenediamine (OPD), substrate was added and the color developed wasdetermined by an ELISA reader at 450 nm.

c. FACS Analysis of Peptide-Inhibition Experiments

BAT mAb biotinylated (20 μg/ml) was incubated with differentconcentrations of the peptide selected from the phage display analysis(0.01-40 μg/ml) overnight at 4° C. Daudi cells (0.5×10⁶) were incubatedwith the antibody or either with the combination of antibody and peptidefor 2 hours on ice. After washing streptavidin FITC was added for 30minutes. Cells were analyzed by a FACScan (Becton Dickinson & Co., NJ,USA).

d. In Vivo Effect of the Peptide Selected from the Phage DisplayAnalysis

C57BL mice were injected with 20 μg of peptide in CFA (complete Freund'sadjuvant) into the footpad. Control mice were injected with CFA alone.After seven days a boost was given with 20 μg of the peptide in PBS. B16melanoma cells (0.5×10⁶ cells/mouse) were inoculated, subcutaneous(s.c.), on day 1 or on day 8 after peptide injection and volume of tumorwas measured every two days. Blood was taken from mice on day 14, 21 and28 after peptide injection for testing specific anti-peptide antibody insera using ELISA method.

Results

a. Isolation of Peptide A (SEQ ID NO 1)

Increased enrichment in the number of plaque forming units (pfu) ofphages which were positive for binding to BAT mAb was observed aftereach panning. Following the 4^(th) panning pfu was 2×10⁴ and after the6^(th) panning a number of 1.5×10⁵ pfu was observed. The number ofphages used in each panning was 1×10⁹. After the sixth panning, 100% ofthe phages were positive for binding to BAT mAb. DNA from 40 positiveclones was sequenced. The 40 phage clones exhibited the amino acidsequence:

-   (Peptide A, SEQ ID NO 1) PRRIKPRKIMLQ    and the nucleic acid sequence:-   (SEQ ID NO 18) CCTCGACGAATAAAGCCCAGGAAGATCATGCTGCAA. Binding of the    phage was highly specific. Moreover, binding to control antibodies    including nonrelated antibody like IgG-3 mAb was not observed.    b. Inhibition of BAT binding to the Selected Phages by the Synthetic    Peptide

To ensure that the interaction between the selected phages and BAT mAbis caused by the insert sequences, peptides encompassing this regionwere synthesized (i.e. Peptide A) and their ability to compete with BATmAb binding to the phage was assessed. Immobilized phage on microtiterELISA plates coated with rabbit anti-phage M13 serum was incubated with6 μg/ml of the antibody. BAT mAb was pre-incubated with increasingamount of synthetic Peptide A. Peptide A inhibited the binding of theantibody in a dose dependent manner with an inhibition concentration(IC₅₀) value of 6.6×10⁻⁷ M (FIG. 1).

c. FACS Analysis of Peptide Inhibition Experiments

BAT mAb were shown to bind a membrane determinant on Daudi cells.Accordingly, the ability of the synthetic Peptide A to inhibit thisbinding was assessed. Biotinylated BAT (20 μg/ml) was pre-incubated withincreased amounts of Peptide A. The mixture was added to Daudi cells andantibody bound was stained with strept-avidin FITC. Peptide A (1 to 40μg/ml) inhibited the binding of BAT mAb to Daudi cells in a dosedependent manner (FIG. 2).

d. In Vivo Anti-Tumor Effect of the Selected Peptides.

C57BL mice (n=4), inoculated s.c. with B16 melanoma cells, wereimmunized with 20 μg of the peptides. Control animals were injected onlywith CFA. The average melanoma tumor volume was found to decrease inmice immunized with the peptides (FIG. 3).

Blood samples were withdrawn from control and immunized mice on day 21and 28 after immunization. Sera from 4 control mice and 4 immunized micewere incubated on microtiter ELISA plates with immobilized peptide.ELISA results indicated that higher concentrations of anti-Peptide Aantibodies were observed in the sera of the immunized mice as comparedwith the sera of control mice (FIG. 4). These results explain the tumorgrowth inhibition in the treated mice (see FIG. 3).

Experiment 2

Materials and Methods

a. Isolation of Epitope-Presenting Phage from a Phage Epitope Library

The phage display peptide library is based on a combinatorial library ofrandom peptide 12-mer fused to a minor coat protein (pIII) of M13 phage.A library sample containing 2×10¹⁰ infectious phage particles wassubjected to 3 rounds of panning. For biopanning selection cycles, 20 μgof biotinylated BAT monoclonal antibody was used. The phage waspre-incubated with the biotinylated antibody at room temperature for 1hour. The reaction mixture was then layered in 1 ml of TBS 0.5% Tween onstrept-avidin coated and blocked 60-mm polystyrene Petri dishes for 30minutes at room temperature. Unbound phages were removed by 10 timeswashings in TBS 0.5% tween. The remaining phages were eluted with 1 ml0.2M Glycine-HCl (pH 2.2), 1 mg/ml BSA. The eluate was neutralized andused to infect E. coli strain ER2537. After each round of panning phagewas titer on LB/IPTG/Xgal plates. After the second cycle of biopanning,the number of phages was amplified. The an-amplified last round wastittered and plaques were used for DNA sequencing.

b. Phage-ELISA

Wells of microtiter plates were coated with 100 μl of a 1:1000 dilution(0.1M NaHCO3, pH 8.6) of rabbit anti-phage M13 serum by incubationovernight at 4° C. Coated plates were washed 3 times with PBS 0.05%Tween. Then, 100 μl of enriched phage clones, containing 10⁹ phageparticles, were then added to the wells and incubated for 1 hour at 37°C. After incubation, wells were blocked with 1% BSA in PBS for 1 hour atroom temperature, washed and incubated with the antibody overnight at 4°C. After washing bound antibody was detected with IgG Peroxidaseconjugated (Fab specific) for 45 minutes. After washing OPD subtract wasadded and the color developed was determined by an ELISA reader at 450nm.

Results

a. Isolation of Peptide B (SEQ ID NO 11) and Peptide C (SEQ ID NO 17)

In view of the anti-tumor effect of BAT mAb, it was aimed to select apeptide that binds specifically the antibody from a 12-mer random phageepitope library. After each panning enrichment in the number of plaqueforming units (pfu) was observed. Resulting from 1^(st) panning a numberof 5.6×10⁶ pfu was obtained, from the 2^(nd) one, 2×10² pfu, afteramplification the number of pfu was 2×10⁸ and after the last round ofbiopanning 1×10³ pfu were counted (Table 1).

TABLE 1 Screening for BAT binding peptide. Panning 1 Panning 2 Panning 3Number of phages  2 × 10¹⁰ 5.6 × 10⁶ 2 × 10⁸ PFU obtained 5.6 × 10⁶   2× 10² 2 × 10³ (sequenced 52) Percent (%) 0.028 0.004 0.0005

DNA from 42 positive clones was sequenced. The DNA from 31 of the phageclones exhibited the sequence:

-   CAGAGGATACTGCAGCAAATTAATCTTCCCAGGATC (SEQ ID NO 28) encoding for a    peptide of SEQ ID NO 11, also termed Peptide B.

The DNA from 3 of the phage clones exhibited the sequence:

-   AACCGAATCAGGACAAATACTAAGCTCATGAACAGC (SEQ ID NO 34) encoding for a    peptide of SEQ ID NO 17, also termed Peptide C.    The rest of the 8 positive clones exhibited different sequences.    b. Phage ELISA on Anti-M13 Plates.

To ensure that the interaction between the selected phages and BAT mAbis caused by the insert sequences, ELISA on anti M13 binding plates wasperformed. The ability of the positive phage to bind BAT mAb (5 μg/ml)was assessed. 1×10⁹ phages from positive pfu were incubated on anti-M13plates and binding to BAT mAb was assessed. ELISA detected only phageswith peptide B in the insert (FIG. 5, phages N51 and N52). Phages withother sequences did not bind to BAT monoclonal antibody (FIG. 5, phagesN40, N6, N7, and N37).

Example 3 In Vivo Effect of Peptides

Materials and Methods:

-   Peptides: Peptide A and Peptide B and variants, are shown in Table 2    (SEQ ID NO 1-17). Peptide were produced by amino-acid synthesis as    known in the art.-   Mice: C57BL female mice, 6-8 weeks old.-   Tumors: B16 syngeneic melanoma cells were inoculated in order to    induce the formation of lung metastases. Cells were inoculated i.v.    on day 1 following the first peptide injection. Metastases were    detected in the lung by their dark coloration, resulting from    melanin production in the cells. Tumor growth was monitored by lung    weight compared to control mice.-   Vaccine composition and dose: A vaccine immunization composed: 10 μg    of a peptide and 100 μl of complete Freund's adjuvant (CFA) or PBS.-   Vaccination Protocol: C57BL mice were injected with the above dose    of peptide vaccine in CFA into the footpads, followed by two boosts    of the above dose in PBS on the 7^(th) and 14^(th) day following the    first immunization. Control mice were injected with vehicle alone.    BAT (10 μg) monoclonal antibody was injected on day 10 post    tumor-inoculation as a positive control. Lung weight was measured on    day 24 post tumor inoculation.    Results:

Peptide A (SEQ ID NO 1) and several of the variants based on thesequence of Peptide A (SEQ ID NOS 6 and 8-10; FIG. 6) and on thesequence of Peptide B (SEQ ID NOS 14 and 16; FIG. 7) exhibited antitumor activity similar to that of BAT monoclonal antibody. Theanti-tumor activity as a measure of lung weight was approximately twicehigher as compared with control (non-treated tumors). Other peptideswere found less effective than BAT monoclonal antibody.

TABLE 2 Peptide analogs Peptide SEQ ID NO Peptide sequence DNA SEQ ID NO 1 (Peptide A) PRRIKPRKIMLQ 18  2 prrikprkimlq-amide 18  3 PRRFKPRKIBLQ19  4 PRRIKPRKIBFQ 20  5 PRRIKPRKIBLQ 21  6 PRRIKARKIMLQ 22  7PRKIKPRKIMLQ 23  8 --RIKPRKIMLQ 24  9 PRRIKPRKIM-- 25 10acetyl-PRRIKPRKIMLQ 18 11 (Peptide B) QRILQQINLPRI 26 12 QRILQQINLARI 2713 QRILQEINLPRI 28 14 QRILQQINLPKI 29 15 --ILQQINLPRI 30 16 QRILQQINLP--31 17 (Peptide C) NRIRTNTKLMNS 32

Example 4 NK and CTL Cytolytic Activity in Peptide-Immunized Mice

Material and Methods

Cytotoxicity was examined by measuring the release of radiolabeledchromium from target cells. Cells (2-4×10⁶ YAC, B16 or 3LL) were labeledwith 200 μCi [⁵¹Cr]-chromate, in serum free media for 1 h at 37° C.Following three washes, cells were resuspended in complete medium andplated at 10⁴ cells/well. Mouse splenocytes were removed on theindicated days (YAC-day 7, B16 and 3LL-day 21) following peptideimmunization. Vaccination regimen was identical to that used intumor-inhibition experiments (see Example 3). Splenocytes were mixedwith target cells at the indicated ratios and incubated for 12 h at 37°C. Supernatants were monitored for radioactivity. Cytotoxicity wasdetermined as follows,% Lysis=100×(Re−Rs)/Rmax−Rs).where Re is the measured release, Rs is the spontaneous release in thepresence of medium alone and Rmax stands for the maximal releaseobtained by incubating target cells with Triton x100. ⁵¹Cr release wasdetermined at effector-to-target cell ratios of 1:5, 1:25 and 1:50.Results

Tumor inhibition by peptide vaccines may depend on both humoral andcellular effects elicited by the immunization. To examine whetherpeptides A and B induce cellular toxicity, mice immunized with thepeptides were studied for their ability to mount tumor-cell lysis invitro. Following an immunization regimen that elicits an anti-tumorresponse, splenocyte cytotoxicity against a variety of tumor cells wasobserved (FIG. 8). Interestingly, this activity was induced against bothNK sensitive cells (YAC; FIG. 8A) as well as NK insensitive cells (B16and 3LL; FIG. 8B and FIG. 8C, respectively), suggesting involvement ofboth populations in the response. Characteristically, NK-dependent lysiscould be observed as early as 7 days following immunization, inaccordance with early stimulation of NK cells. NK-independent lysis ofB16 and 3LL cells followed a classical time-response of maximal efficacyaround day 21.

The above effects could be induced to a similar extent by both peptidesA and B, reaching an impressive activity of up to % 80 lysis. Peptide N,a non-relevant control peptide could not elicit a similar response, inaccordance with its inability to inhibit tumor growth.

Example 5 Anti-Peptide A and B Antibodies Compete with BAT MonoclonalAntibody Binding to Daudi Cells

Materials and Methods:

a. Antibody Purification:

To examine the ability of BAT-selected peptide-vaccines to elicitBAT-like antibodies, serum from peptide-immunized mice was collected andpurified on protein-G sepharose beads according to manufacturer'sinstructions (Pharmacia Corp. NJ, USA).

b. Inhibition of BAT Binding:

Daudi cells (0.5×10⁶) were pre-incubated with anti-peptide purifiedserum, 5, 10 and 20 μg/ml of each purified antibody, for 2 hours on ice.Biotinylated BAT monoclonal antibody (40 μg/ml) was added and cells wereincubated for an additional 2 h period at 4° C. Following extensivewashing streptavidin FITC was added for 30 minutes. Cells were analyzedby FACScan (Becton Dickinson & Co., NJ, USA). Binding of anti-peptideantibodies alone (20 μg/ml) was determined in a similar manner, however,the detection was performed by incubation with a secondary antibodyagainst mouse IgG conjugated to FITC. In a control sample, cells wereexposed only to the secondary antibody.

Results

It is plausible that peptides mimicking the antigenic site of BATmonoclonal antibody can elicit production of antibodies in the serumwith similar binding characteristics to those of the original BAT. Toexamine this possibility, antibodies purified from the serum ofpeptide-immunized mice were studied for their ability to inhibit thebinding of BAT to its antigen (FIG. 9). The binding of anti-A and anti-Bantibodies to Daudi cells was verified (FIG. 9B) with respect to control(FIG. 9A), suggesting recognition of BAT antigen.

Determination of BAT binding in the presence of anti-A (FIG. 9C) andanti-B (FIG. 9D) antibodies revealed a common epitope. Both peptidesinduced mouse antibodies capable of significantly inhibiting BAT bindingto its target cells, as demonstrated by the decrease in fluorescentlabeling by BAT. This observation is explained by the fact that in thepresence of the purified anti-peptide antibodies, BAT antibody couldoccupy less antigenic determinants on the surface of antigen expressingcells.

Example 6 Anti-Peptide A and B Antibodies Induce Human LymphocyteProliferation

Materials and Methods:

a. Human-PBL (Peripheral Blood Lymphocytes) Preparation

Human normal donor blood was diluted 1:1 in PBS. Histopaque (1077-1SIGMA) was added (1:2 v/v) and the solution was centrifuged for 30 min,1600 rpm at 4° C. The lymphocyte inter-phase ring was collected, cellswashed and incubated in complete media for one hour at 37° C. to removeadherent cells. PBL were collected from supernatant.

b. Thymidine Incorporation

PBL were dispensed at a concentration of 2×10⁶ cells/ml (200 μl) in96-well flat-bottom plates in complete medium. BAT or anti-peptideantibodies (1 μg/ml) were added for 5 days followed by incubation in thepresence of ³[H]Thymidine (1 μCi/well) for 16 h at 37° C. Cells wereharvested and radioactivity determined using a liquid B-scintillationcounter.

Results

Human PBL incubated in the presence of antibodies purified frompeptide-immunized mice, demonstrated an increased proliferation, similarto that induced by BAT (FIG. 10). These antibodies were effective at asimilar concentration and time interval as those of BAT antibody,suggesting a resemblance in efficacy and may be in mechanism. Theresults suggest that lymphocyte stimulation is correlated with theability of the vaccine of the invention to elicit an anti-tumor effect.The results further indicate that the antigenic epitope recognized byBAT is involved in lymphocyte activation.

The ability of the peptide-induced antibodies to stimulate lymphocyteproliferation is, most probably, the mechanism underlying their abilityto trigger anti-tumor cytotoxicity (as shown in Example 4). In thesetting of disease, anti-cancer lymphocytes that are present but beneatha threshold require an efficient immune-response such as the stimulationthat is represented by the peptide of the invention in the presentexample.

Example 7 Peptide Vaccination in Cancer Patients

Vaccination of human subjects with peptides recognized by BAT monoclonalantibody is tested in several studies. The target population for thesestudies is, in the first case, cancer patients. In these patients, thevaccine is expected to elicit an anti-tumor immune response, allowing toalleviate, reduce, cure or at least partially arrest the disease.

-   Phase I—Safety study: Double-blind, rising dose, Placebo-controlled.-   Phase II—Safety & Efficacy study: Double-blind, rising dose,    Placebo-controlled.

Patients who sign informed consent and fulfill inclusion criteria arerandomized in a 1:1 ratio to receive the peptide vaccine or placebo(vehicle or adjuvant only). Peptide vaccine and placebo are diluted insterile saline to generate the administered dosage form. The medicationis administered by a suitable route of administration, for example, afast drip intravenous infusion or peristaltic pump or subcutaneously.

The primary efficacy endpoints of the trial are: the reduction in tumorsize, NK and CTL activation, proliferation of lymphocytes and humoraland cellular immune responses to tumor antigens, evaluation of peptideconcentration in the blood (pharmacokinetics).

Treatment of cancer patients may exclusively include the peptide vaccineor may include another vaccine comprising a BAT monoclonal antibody. Inthe latter type of treatment the two vaccines may be administeredseparately at similar time points or at different time points of thetreatment. Safety is assessed by comparing the rate of adverse events,classified according to body system, severity and relation to treatment,between the two groups.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention. Thusthe expressions “means to . . . ” and “means for . . . ”, or any methodstep language, as may be found in the specification above and/or in theclaims below, followed by a functional statement, are intended to defineand cover whatever structural, physical, chemical or electrical elementor structure, or whatever method step, which may now or in the futureexist which carries out the recited function, whether or not preciselyequivalent to the embodiment or embodiments disclosed in thespecification above, i.e., other means or steps for carrying out thesame functions can be used; and it is intended that such expressions begiven their broadest interpretation.

1. An isolated peptide comprising at least one epitope that isrecognized by and binds to a BAT monoclonal antibody, said peptideselected from the group consisting of: (a) a peptide having the sequenceof any one of SEQ ID NOs: 3 through 5; (b) a peptide having at least 90%identity with a peptide of (a); and (c) a combination of peptidesaccording to (a) or (b).
 2. The peptide according to claim 1, whereinthe peptide has SEQ ID NO
 3. 3. The peptide according to claim 1,wherein the peptide has SEQ ID NO
 4. 4. The peptide according to claim1, wherein the peptide has SEQ ID NO
 5. 5. An immunomodulating agentcomprising the peptide of claim
 1. 6. An immunomodulatory vaccinecomprising at least one peptide according to claim 1 and apharmaceutically acceptable adjuvant.
 7. The vaccine according to claim6, wherein the adjuvant is selected from the group consisting of analuminum salt and an oil in water emulsion.
 8. A diagnostic agent fordetecting cancer comprising a peptide according to claim
 1. 9. Apharmaceutical composition comprising as an active ingredient at leastone isolated peptide comprising at least one epitope that is recognizedby and binds to a BAT monoclonal antibody, said peptide selected fromthe group consisting of: (a) a peptide having the sequence of any one ofSEQ ID NOs: 3 through 5; (b) a peptide having at least 80% identity witha peptide of (a); (c) a combination of peptides according to (a) or (b);and a pharmaceutically acceptable carrier, excipient or diluent selectedfrom the group consisting of: glucose, mannitol, inositol, sucrose,lactose, fructose, starch, cornstarch, microcrystalline cellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, orpolyvinylpyrrolidone.
 10. The pharmaceutical composition according toclaim 9, further comprising one or more of a preservative, a thickener,or a biodegradable polymer.
 11. The pharmaceutical composition accordingto claim 10, wherein the thickener is selected from the group consistingof: a natural gum, a cellulose derivative, or an acrylic or vinylpolymer, and the biodegradable polymer is poly-1,4-butylene succinate,poly-2,3-butylene succinate, poly-1,4-butylene fumarate orpoly-2,3-butylene succinate.
 12. The pharmaceutical compositionaccording to claim 9, wherein the peptide is present as the pamoate,tannate, stearate or palmitate salt thereof.
 13. A method for treatingcancer in a subject in need thereof comprising the step of administeringa therapeutically effective amount of a composition according to claim9.
 14. A method for treating cancer in a subject in need thereofcomprising the step of administering a therapeutically effective amountof a peptide according to claim 1.